Chitinase cDNA clone from a disease resistant american elm tree

ABSTRACT

cDNA as set forth in SEQ ID NO:1 encoding a chitinase, a signal peptide and a vacuole sorting peptide. The chitinase is antifungal and can be used to treat plants, particularly American elms infected with a fungus which kills the trees. The cDNA can be used to produce recombinant cells which produce the chitinase or which impart resistance to the fungi.

This is a divisional of application(s) Ser. No. 08/286,020 filed on Aug.04, 1994; now U.S. Pat. No. 5,539,095.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

The present invention relates to a unique cDNA derived from a diseaseresistant hybrid Elm Tree which encodes a chitinase, a signal peptideand a sorting peptide. In particular the present invention relates tocDNA as set forth in SEQ ID NO:1.

(2) Description of Related Art

Chitinases are pathogenesis response-related proteins found in a widevariety of plants. Several lines of evidence suggest strongly thatchitinases are antifungal proteins. Accumulation of chitinase (Meins,F., Ahl, P., Plant Cell 61:155-161 (1989); Metraux, J. P., et al.,Physiol Mol Plant Path 28:161-169 (1986); and Rasmussen, U., et al.,Planta 187:328-334 (1992)) and mRNA encoding chitinase (Roby, D., etal., Plant Sci 52:175-185 (1987); Roby, D., et al., Physiological andMolecular Plant Pathology 33:409-417 (1988); and Meins, F., et al.,Plant Cell 1:447-457 (1989)) are induced strongly during the course of afungal infection. This induction is also seen when plant tissue istreated with fungal cell wall material (Kurosaki, F., et al., PhysiolMol Plant Path 31:211-216 (1987); Roby, D., et al., Biochem Biophys ResComm 143(3):885-892 (1987)). Ethylene, a gaseous plant hormone alsoproduced during the course of a fungal infection, strongly induceschitinase activity (Boller, T., Oxford Surveys of Plant Molecular andCell Biology 5:145-174 (1988); and Boller, T., et al., Planta 157:22-31(1983)). Finally, induction also results from wounding (Parsons, T. J.,et al., Proc Natl Acad Sci USA 86:7895-7899 (1989)), conditions whichcould mimic cell death during an active infection.

WO 90/07001 to Broglie et al describes DNA encoding chitinases for usein inhibiting fungi in detail. The techniques described in this patentapplication are applicable to the present invention.

In elms, pathogenesis of the fungus Ophiostoma ulmi, the causative agentof Dutch elm disease (DED) is well understood as is the physiologicalresponse of the elm to infection. Very little, however, is known aboutthe molecular mechanisms of elms' disease response, and nothing of theproduction of pathogenesis-related (PR) proteins, such as chitinase.Little is also known about what enables some species of elm to resist O.ulmi infection.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide a novelcDNA from a DED resistant hybrid elm which encodes a chitinase proteinlinked to a sorting peptide sequence and a signal peptide sequence.Further, it is an object of the present invention to provide cellsproducing the chitinase activity. Further still, it is an object of thepresent invention to provide a method and compositions for theapplication of the peptide. These and other objects will becomeincreasingly apparent by reference to the following description and thedrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a nucleotide and deduced protein sequence of the cDNA for theelm pHS2 clone. Underlined residues in the N-terminus represent thesignal peptide. Underlined residues in the C-terminal end represent theC-terminal extension. Underlined residues in the 3' untranslated regionindicate putative polyadenylation signals.

FIG. 2 is a chart showing a comparison of pHS2 predicted proteinsequence to that of Populus WIN6 (Parsons, T. J., et al., Proc Natl AcadSci USA 86:7895-7899 (1989)), bean pCH18 (Broglie, et al., Proc NatlAcad Sci USA 83:6820-6824 (1986)) and tobacco CHN17 (TOB (A); (Shinshi,H., et al., Plant Mol Biol 14:357-368 (1990)) chitinases. Only residuesdiffering from that from pHS2 are shown. Dashes to left of pCH18 and TOB(A) substitute for the signal peptide. Dashes to the left of WIN6indicate limits of published sequence. Dashes within sequences indicaterealignment for greater sequence identity. ← indicates C-terminus ofpredicted protein product. Sequence alignment aided by Seqed™ for theMacintosh.

FIG. 3 is a graph showing predictions of α-helix, β-sheet, andturn-forming regions by the Chou-Fasman (CF) and Robson-Garnier (RG)algorithms. A consensus of the two methods is also calculated (CfRg Hlx,Sht, Trn). Analysis performed by MacVector™ software (InternationalBiotechnologies, Inc., Eastman Kodak Co., Rochester, N.Y.

FIGS. 4A-4C are graphs showing predictions of ECH2 hydrophilicity usingthe Hopp-Wood, Kyle-Doolittle, and GES algorithms. Window size=7 in allcases. The analysis was performed by MacVector™ software.

FIGS. 5A and 5B are graphs. FIG. 5A shows the antigenic index of ECH2.FIG. 5B shows the surface probability of ECH2. Analysis performed byMacVector™ software.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention relates to a gene which encodes a polypeptide inpure form having a chitinase-like activity and a signal and a sortingpeptide as set forth in SEQ ID NO:1.

Further, the present invention relates to a composition which comprisesa purified protein which inhibits Ophiostoma ulmi and has achitinase-like activity encoded by a gene as set forth in SEQ ID NO:1and an agriculturally acceptable carrier, the composition containing anamount of the protein which inhibits the Ophiostoma ulmi uponapplication to a plant.

The present invention also relates to cells transformed with the gene.

Finally, the present invention relates to a method for inhibiting afungus which comprises applying a protein having a chitinase-likeactivity and encoded by a gene as set forth in SEQ ID NO:1 in aninhibiting amount to the fungus.

The present invention includes transformed cells which produce thechitinase, either separately or with the sorting and signal peptides.One preferred cell is Escherichia coli. The DNA is introduced into thecells by known methods. The cells can be in culture to produce thechitinase or can be in the form of plants which are resistant toOphiostoma ulmi, particularly elms.

The hydrolytic enzyme chitinase has been implicated in the resistance ofmany plants to fungal attack, primarily because it accumulates to highlevels during infection or in the presence of fungal cell wall material.Although many chitinases have been described in herbaceous plants, thestudy of these enzymes in hardwoods has been extremely limited. Theisolation and initial characterization of a 1225 bp full-length cDNAclone encoding a chitinase from a DED "resistant" selection NPS 3-487hybrid elm Ulmus spp. was unexpected.

The cloning was based upon sequence homology using WIN6, a cDNA to awound-induced mRNA from poplar. WIN6 was used to probe a cDNA library of3-487 mRNA from callus tissue induced with Ophiostoma ulmi, thecausative organism of Dutch elm disease. The predicted translationproduct is a 321 residue Class I chitinase (by the nomenclature ofShinshi et al., Plant Mol Biol 14:357-368 (1990)) containing thecharacteristic catalytic and cysteine-rich domains. A C-terminalextension, which has been shown to route proteins into the vacuole, isalso present. This chitinase has 70.5% sequence identity with Class I Atobacco chitinase, 68.6% with the bean pCH18 translation product, and67% with that from poplar WIN6. The cDNA sequence and predictedtranslation product, as well as several sequence analyses, are presentedherein.

EXAMPLE 1

A chitinase-like cDNA sequence was isolated from an O. umli-induced NPS3-487 cDNA library. NPS-3-487 is an American elm selected by theNational Park Service in Washington, D.C. and was found to be moderatelyresistant to the DED using inoculation techniques.

Materials and Methods

Callus cultures were initiated from young leaves of greenhouse grownrooted cuttings of hybrid elm selection NPS 3-487. First and secondexpanded young leaves were surface sterilized in 20% CLOROX (CloroxProfessional Co., Oakland, Calif.) hypochlorite and cultured in elmcallus initiation medium including 1/2 strength modified MS medium, 40g/L maltose, 100 mg/L casein hydrolysate, 100 mg/L thiamine, 2.5 mM 2,4D, 1 mM kinetin, 100 ml/L coconut milk (Sigma Chemicals, St. Louis, Mo.)and 6.5 g/L Phytagar (BRL). Cultures were incubated in dark at 24°±2° C.for 3-4 weeks or until distinct calli (4-5 mm diameter) wereestablished. Vigorously growing calli were subcultured on the same mediaevery three to four weeks.

Three avirulent (Q311, Q412 and SSMF) and one virulent (CEF16K) strainsof O. ulmi (described in the literature) were used for putativeinduction of elm callus. Fungi were grown of PDA (potato dextrose agar,Sigma Chemicals)-impregnated filter paper circles for three to fourdays; filters containing the pathogen were then overlaid on calluscultures for 24 hours. Fungus-treated calli were flash frozen in liquidnitrogen and stored at -80° C. until used.

A unidirectional cDNA library was custom made by Clontech (Palo Alto,Calif.) as directed by the inventors. Briefly, total RNA was isolatedfrom equal mixtures of calli induced as above, using a guanidium-basedmethod and poly A+mRNA affinity purified via poly U sepharose usingstandard protocols (J. Sambrook, E. F. Fritch and T. Maniatis, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, N.Y.(1989)). Olido dT (septadecamer) attached to an Xho I linker was used asthe primer for first strand synthesis. Double stranded cDNA as linkedwith Eco RI adapters and the complete, end modified cDNA cloned in EcoRI/Xho I-opened. bacteriophage lambda Zap II (Stratagene). Therecombinant phage was plated on E. coli SURE (SURE strain:e14-(mcrA),Δ(mcrCB-hsdSMR-mrr) 171, endA1, supE44, thi1, gyrA96, relA1, lac, recB,recJ, sbcC, umuC: Tn5 (kan^(r)), uvrC, (F' proAB, lacI^(q) ZΔM15,Tn10(tet^(r))) (Stratagene, San Diego, Calif.) cells and amplified onecycle.

A 764 bp wound inducible Populus spp (poplar) cDNA (Parsons, T. J., etal., Proc Natl Acad Sci USA 86:7895-7899 (1989)), which appears toencode a chitinase, was used as a heterologous probe at moderatestringency (3X SSPE at 60° C.) to screen the cDNA library. This yielded0.2% primary plaques with varying signal intensities. One of the tripleplaque purified phage clones was subcloned in pBS II KS using biologicalrescue and sequenced at the Plant Research Laboratory SequencingFacility at Michigan State University.

Sequence analyses of the predicted protein product of pHS2 wereconducted on a Macintosh IIsi (Apple Computer Co., Cupertino, Calif.)with either the DNA Strider™ (shareware version (InternationalBiotechnologies Inc., Eastman Kodak Co., Rochester, N.Y.) or MacVector™software packages (International Biotechnologies Inc., Eastman KodakCo., Rochester, N.Y.). Comparisons to known chitinase sequences wereperformed by hand aided by Seqed for the Macintosh.

The full length cDNA clone, designated pHS2, has a 951 nucleotide openreading frame (FIG. 1). Using the 317 amino acid deduced sequence as thereading frame, a search of the PDB (protein DataBank) Swissport, PIRGenbept (cds translation from Genbank), Kabartpro (Kabartsev of ProteinJ. immunological interest of value, Genbank/EMBL databases using BLAST(Basic Local Alignment Search Tool) The National Center of BiotechnologyInformation NIH, Bethesda, Md. (Kardin, S. and Altschul, S. F., Proc.Natl. Acad. Sci. USA 87:2264-2268 (1990)) showed 50 of the 53 bestmatches to be chitinases; the remaining three were lectins oragglutinins, which share a chitin-binding domain with the pHS2translation product. The predicted amino acid sequence of this 1236 bpclone, designated ECH2, revealed homology to bean, poplar, and tobaccoclass I chitinases (FIG. 2) strongly suggesting that this cDNA encodes achitinase in this hybrid elm. The amino acid sequence of thischitinase-like product has 68.6% sequence identity to that of thetranslation product of the Phaseolus chitinase clone bean pCH18(Broglie, et al, Proc natl Acad Sci USA 83:6820-6824 (1986)), 70.5% tothe tobacco CHN17 chitinase (Shinshi, H., et al., Plant Mol Biol14:357-368 (1990)), and 67% to poplar WIN6 (Parsons, et al., Proc NatlAcad Sci USA 86:7895-7899 (1989)) translation product (Comparisons donot consider the signal peptide sequences).

The first 21 residues of pHS2 comprise a signal peptide (FIG. 1); thispeptide has the characteristic hydrophobicity coupled with a positivelycharged residue within three residues of the initial methionine (vonHeijne, G., Eur J Biochem 133:17-21 (1983)). The C-terminal sevenresidues appear to comprise a second routing peptide known as aC-terminal extension. It has been established that this sequence issufficient and necessary to direct a protein into the vacuole(Chrispeels and Raikhel, The Plant Cell 3:1-9 (1991)) .

The 3' untranslated region pHS2 reveals three putative polyadenylationsignals as described by Joshi (Joshi, C. P., Nucleic Acid Res. 15(23):9627-9640 (1987)). A classic AATAAA signal is not present; however, anAATAAG and an AACAAAare present. Currently it is believed that theAATAAG is the more probable signal as this motif is present in anotherchitinase-like clone from this elm. Immediately 5' to the AATAAG is aYAYTG-like sequence (CAATG). Finally, ten nucleotides downstream, aTGTGTGCGCT is present which has high identity to a third olyadenylationmotif (Joshi, 1987).

Analysis of Predicted Protein Product

The predicted translation product of pHS2 was analyzed for surfaceprobability, the presence of hydrophobic domains, alpha helices and betasheets, and antigenic sites. Using a consensus of the Chou-Fasman andRobson-Garnier algorithms (FIG. 3), ECH2 contains ten regions with ahigh probability of forming β-sheets. No alpha helices are predicted;although three regions are predicted by the Chou-Fasman method, none areby Robson-Garnier. 28 regions which may form turns are also predicted.It should be noted that, of the two prediction algorithms,Robson-Garnier is the more stringent (von Heijne, G., Sequence analysisin molecular biology: Treasure trove or trivial pursuit. Academic Press,Inc. San Diego (1987)). The overall hydrophilicity of ECH2 wasdetermined by the Hopp-Woods (HW; FIG. 4A; Hopp and Woods, Proc NatlAcad Sci USA 78(6):3824-3828 (1981); Kyte-Doolittle (KD; FIG. 4B; Kyteand Doolittle, J. Mol Biol 157:105-132 (1982), and GES (FIG. 4C)algorithms. While the HW method predicts a somewhat hydrophobic protein,the KD and GES methods predict a moderately hydrophilic protein. Tworegions predicted as strongly hydrophobic by all three methods includethe signal peptide and the chitin-binding domain. While there are a fairnumber of predicted antigenic sites (FIG. 5A), there are only six majorregions, plus several minor ones, predicted to comprise to the protein'ssurface (FIG. 5B). This is interesting in that antigenicity predictionsare largely based on which residues are most likely to be exposed to theprotein's surface.

Certain aspects of the present invention are described in Dutch ElmDisease Research, pages 1-5, published Aug. 9, 1994 by Springer-Verlag,New York, N.Y. It is intended that the foregoing description be onlyillustrative of the present invention and that the present invention belimited only to the hereinafter appended claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 1                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1225                                                              (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iii) HYPOTHETICAL: No                                                        (iv) ANTI-SENSE: No                                                           (v) FRAGMENT TYPE:                                                            (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Ulmus Americana                                                 (B) STRAIN: NPS 3-487                                                         (C) INDIVIDUAL ISOLATE: N/A                                                   (D) DEVELOPMENTAL STAGE: N/A                                                  (E) HAPLOTYPE: N/A                                                            (F) TISSUE TYPE: N/A                                                          (G) CELL TYPE: N/A                                                            (H) CELL LINE: N/A                                                            (I) ORGANELLE: N/A                                                            (vii) IMMEDIATE SOURCE: N/A                                                   (viii) POSITION IN GENOME: N/A                                                (ix) FEATURE:                                                                 (A) NAME/KEY: chitinase encoding DNA                                          (B) LOCATION:                                                                 (C) IDENTIFICATION METHOD: sequencing                                         (D) OTHER INFORMATION: DNA needed for chitinase                               in elm.                                                                       (x) PUBLICATION INFORMATION:                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       CGGGATTAACGAGATTCCCACTGTCCCTGTCTACTACCCAGCGAAACCACAGCCAAGGGA60                ACGGGCTTGGCAGAATCAGAACTAAACATGAGGTTTTGGGCATTGACGACACTTTCT                     MetArgPheTrpAlaLeuThrThrLeuSer                                                510                                                                           CTT12                                                                         Leu                                                                           CTATTCTCCATTATCCAAGGAGGCTGGGCAGAGCAATGTGGAAGCCAAGCT                           LeuLeuSerIleIleGlnGlyGlyTrpAlaGluGlnCysGlySerGlnAla                           152025                                                                        GGGGGTGCA180                                                                  GlyGlyAla                                                                     30                                                                            GTCTCTCCCGTTGGGCTCTGCTGCAGCAAATTTGGGTGGTGTGGGAGCACA                           ValCysProValClyLeuCysCysSerLysPheGlyTrpCysGlySerThr                           354045                                                                        AACGAGTAC240                                                                  AsnGluTyr                                                                     50                                                                            TGTGGTGATGGCTGCCAAAGCCAATGTGGCGGCAGCGGTAGCGATGACATT                           CysGlyAspGlyCysGlnSerGlnCysGlyGlySerGlySerAspAspIle                           556065                                                                        GGCGGTCTC100                                                                  GlyGlyLeu                                                                     70                                                                            ATATCAAGCTCCGCCTTTAATGACATGCTTAAGCATCGTAACGACGGTGGT                           IleSerSerSerAlaPheAsnAspMetLeuLysHisArgAsnAspGlyGly                           758085                                                                        TGTCCTGCC360                                                                  CysProAla                                                                     90                                                                            AAGGGGTTTTACACCTATGATGCTTTTATTGCGGCTGCCAAGGCTTTCCCT                           LysGlyPheTyrThrTyrAspAlaPheIleAlaAlaAlaLysAlaPhePro                           95100105110                                                                   GCATTTGGC420                                                                  AlaPheGly                                                                     TCCACCGGCGATGATACCACCCGTAAAAGGGAGATTGCSGCTTTCTTAGGT                           SerThrGlyAspAspThrThrArgLysArgGluIleAlaAlaPheLeuGly                           115120125                                                                     CAAACTTCC480                                                                  GlnThrSer                                                                     130                                                                           CATGAAACTACAGGTGGGTGGGCAAGTGCACCCGACGGTCCATACTCTTGG                           HisGluThrThrGlyGlyTrpAlaSerAlaProAspGlyProTyrSerTrp                           135140145                                                                     GGATACTGC540                                                                  GlyTyrCys                                                                     150                                                                           TACAATAGGGAGCAAAACCCTTCTTCCGATTATTGTTCTTTTAGTCCTACT                           TyrAsnArgGluGlnAsnProSerSerAspTyrCysSerPheSerProThr                           155160165                                                                     TGGCCTTGT600                                                                  TrpProCys                                                                     170                                                                           GCTTCCGGAAAGAGATACTTTGGCCGTGGTCCCATTCAACTCTCCTGGAAC                           AlaSerGlyLysArgTyrPheGlyArgGlyProIleGlnLeuSerTrpAsn                           175180185                                                                     TACAACTAT660                                                                  TyrAsnTyr                                                                     190                                                                           GGACAGTGTGGAAGGGCCATAGGAGCAAACCTATTAAACAACCCTGATCTC                           GlyGlnCysGlyArgAlaIleGlyAlaAsnLeuLeuAsnAsnProAspLeu                           195200205                                                                     GTAGCAACT720                                                                  ValAlaThr                                                                     210                                                                           GACCCTGTCATTTCCTTCAAAACGGCCTTATGGTTCTGGATGACCCCACAG                           AspProValIleSerPheLysThrAlaLeuTrpPheTrpMetThrProGln                           215,220225                                                                    TCACCAAAG780                                                                  SerProLys                                                                     230                                                                           CCCTCGTGCCATGACGTCATCACCGGAAGATGGAGTCCTTCCGGCACCGAC                           ProSerCysHisAspValIleThrGlyArgTrpSerProSerGlyThrAsp                           235240245                                                                     CAGTCGGCC840                                                                  GlnSerAla                                                                     250                                                                           GGCCGAGTTGCGGGCTACGGCGTGATCACCAACATTATCAACGGTGGGATA                           GlyArgValAlaGlyTyrGlyValIleThrAsnIleIleAsnGlyGlyIle                           255260265                                                                     GAATGCGGG900                                                                  GluCysGly                                                                     270                                                                           AAAGGTCAGGTTCCTCAGGTGGTGGATCGGATTGGATTCTACAAGAGGTAC                           LysGlyGlnValProGlnValValAspArgIleGlyPheTyrLysArgTyr                           275280285                                                                     TGTGATATC960                                                                  CysAspIle                                                                     290                                                                           CTTAGAGTTGGCTATGGGAACAATCTTGATTGCTATAACCAGAGGCCTTTT                           LeuArgValGlyTyrGlyAsnAsnLeuAspCysTyrAsnGlnArgProPhe                           295300305                                                                     GGGAATGGA1020                                                                 GlyAsnGly                                                                     310                                                                           CTCTTGTTGGACGCCATGTAACGACTTGTCGTAGTTGTTATACATATCTGT                           LeuLeuLeuAspAlaMet                                                            315                                                                           GTTTTGAGG1080                                                                 GTTTGGCCGTCGTTTGTCGTCACTTCTTCGGTGACAATTTCGTTGTGTTATAGTTACTAT1140              ATATGTCAATGGAATAAGAACAAAGGATGTGTGCACTACAACAAAAAAAAAAAAAAAAAA1200              AAAAAAAAAAAAAAAAAAAAAAAAA1225                                                 __________________________________________________________________________

I claim:
 1. A purified protein having a chitinase activity encoded froma gene as set forth in SEQ ID NO:1 which inhibits Ophiostoma ulmi.
 2. Acomposition which comprises a purified protein which inhibits Ophiostomaulmi and has chitinase activity encoded by a gene as set forth in SEQ IDNO:1 and an agriculturally acceptable carrier, the compositioncontaining an amount of the protein which inhibits the Ophirstoma ulmiupon application to a plant.
 3. A method for inhibiting a fungus whichcomprises applying a protein having chitinase activity and encoded by agene as set forth in SEQ ID NO:1 in an inhibiting amount to the fungus.4. The method of claim 3 wherein the fungus is Ophiostoma ulmi.